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Parkinsons disease is the second most common neurodegenerative disorder that affects the 2 of the population. There is an need for developing a biomarker used to predict the progression of the disease and to improve the diagnosis. In this study various biomarkers are compared on their ability as an assay, where protein based biomarkers in CSF (proteomic approaches) pretends to be an early biomarker of Parkinson's disease.
Parkinson's disease is the common neurodegenerative disorder, affecting 2% of the population among people aging above 65 years and is the second most common disorder after Alzheimer's disease (Thomas et al. 2007). Number of populations may double within the next decades according to the recent projects of epidemiology (Dorsey et al. 2007).
Despite the extensive research on Parkinson's disease, mechanisms underlying are not firmly established due to poor diagnostic, lack of valid biomarkers and therapeutic interventions which are difficult to elaborate resulting in progression of Parkinson's disease.
However developing a validated biomarker benefits to people affected by Parkinson's disease. Apart from this, biomarkers have clinical importance in research for identifying novel neuroprotectatants and surrogate markers have potential application in predicting and measuring the level of progression in disease and in therapeutics intervention responses to pathological and biochemical effects.
Since from past decades to recent studies, development of biomarkers for Parkinson's disease was only focused on imaging techniques which were based on measuring substantia nigra and its loss of dopaminergic neurons such as imaging dopamine transporter (DAT). In addition to this technique , genetic level understandings has lead to the identification of causative genes that can be useful in molecular diagnosis and recent advances and availability of bioinformatics tools made possible in developing fluid biomarkers practicable , cheaper and obtainable blood, cerebrospinal fluid tests may influence upon diagnosis and research in Parkinson's disease. In this study, current biomarkers potent ability and their usefulness in clinical practices, research and their limitations will be discussed. Modern technologies of proteomics which are better approach as a tool to pathogenesis of Parkinson's disease will be discussed in detail.
Apart from motor symptoms , there are other clinical markers associated to Parkinson's disease , such as sleep disorder identification, olfactory discrimination , dysfunction of gastro intestine and cardiac autonomy .but these clinical markers /symptoms are also common in other neurological disorders such as alziehemers disease. Hence they lack specificity and are not precise in predicting the later occurrence of the disease.
In addition to clinical markers, there are genetic as well as genomic approaches to develop diagnosed biomarkers in Parkinson's disease, but genomic analysis holds good as a trait instead as a biomarker (Gasser, 2009). Genetic testing is useful only in predicting the genes which have been identified so far in minority of cases.
Recently, neuronal analysis and plasma or CSF markers are widely accepted and proteomic approaches have potential applications in developing biomarkers to diagnostic Parkinson's disease.
Proteomic approach in Parkinson's disease
Parkinson's disease is characterized mainly a by severe motor symptom which includes slowness of movement, rigidity and uncontrollable tremor (Lang et al. 1998; Eriksen et al. 2005). According to neuropathology Parkinson's disease is characterized as substantia nigra pars compacta (SNpc) contains neurons of neuromelanin (NM), as a loss of dopaminergic and also as an occurrence of lewy bodies (LB) (Lewy et al. 1912).
Fig1: Levels of dopamine in Parkinson's affected and normal neurons.
Neurons which are more pigmented degenerate first suggesting that random depletions of NM does not occur (Hirsch et al. 1988). LB composed of α-synuclein which is small protein consisting of 140 amino acids unfolded state in a solution confirms of vesicles of lipid β-plated sheet in fibrils (Goedert 2001).
Apart from sporadic forms, there are many other familial forms of disease in Parkinson's disease such as mutations of α-synuclein gene. In Parkinsonism, autosomal recessive forms are mutations linked to parkin (PARK3) DJ-1 (PARK 7) and PINK1 (PARK6) genes which are as early onsets of Parkinson's disease. These mutated genes are identified as a dominant of for autosomal forms; α-synuclein, CHL1 (ubiquitin carboxyl terminal hydrolase L1) and LRRK2 (Leucine rich repeat Kinase 2) plays a role in intracellular signaling pathway encoding protein kinase domain. These mutations affect the biochemical process such as oxidative stress (PINK1, DJ-1), aggregation of protein (Parkin, UCHL1, α-synuclein) and intracellular signaling (LLRK2, DJ-1).
Proteomics of Parkinson's disease: Cellular Model
Molecules that act like endogenous toxins are produced by the metabolized DA, when dopaminergic neurons are exposed to various oxidative stresses (Lotharius et al. 2002; Bonifati et al. 2004). Based on this aspect several cell models were developed for DA toxicity.
Often used cellular modeling for neurodegenerative research is human neuroblastoma cell line (SH-SY). This model is used, as it expresses DA transporter and its receptors and also capable of forming storage vesicles, but DA concentration of cytoplasmic SHSY5Y in culture medium has to be raised by extracellular administration.
Proteomics of Parkinson's disease: Animal Model
Using proteomics, currently there are few animals models examined for studies of Parkinson's disease. 2DE differential proteome used to study dysfunction of mitochondrial and ventral midbrain. Usually oxidative damage in parkin deficient mice results in detecting 13 down regulated proteins (Palacino et al. 2004). Neurotoxins used to develop Parkinson's disease in various species, and animal models were tested based on age differences with specific levels of carbonyl, and protein levels were examined which resulted in increased expression levels in Parkinson's disease (Poon et al. 2006). By using MALDI MS imaging and 2DE analysis, expression of protein profiles and their differences in regulation can be spotted.
The overview of protein identification from tissue with Parkinson's disease and the control samples are as shown in fig2
Fig2: Proteomic analysis of 2D-PAGE in disease (PD) and control sample
Proteomics of Parkinson's disease: Humans
Recently, samples from patients affected by neurological disorders are of huge interest as they show the abnormalities which are directly linked to study two types of samples:
Tissue from brain during autopsy.
Cerebrospinal fluid (CSF) sample collected from living patients.
Protein profiling: Brain tissue
Brain sample have advantage of assessing neuropathological examination and provides diagnosis of Parkinson's disease. Particular region of interest can be dissected e.g. LC, SNpc and cerebral cortex, thereby increasing the specificity of study. Major drawbacks of this approach are the contamination, recently brain tissue samples of mouse and humans are used in order to overcome the problem (Crecelius et al. 2008; Hunsucker et al. 2008) by increasing the exposure of post-mortem intervals.
Werner et al., using the same approach in study of SNpc identified 221 spots that differentially expressed in Parkinson's disease groups to that of the controls.
Protein profiling: CSF
CSF is the ideal source for diagnosis and in predicting therapeutic biomarkers due to its closeness to brain structures which undergo degeneration by releasing pathogenic molecules. Added advantage of CSF is that it is available for lifetime of a patient and changes in protein profile can be studied during its entire course of disease. Lumbar puncture (LP) is considered now a day's which can be repeated several times in the same individual.
CSF proteomic approaches have proved their success in case of AD (Zellner et al. 2009) and also in Geutzfeldt-Jakob disease (Brechlin et al. 2008) CSF-2DE maps and other separation methods (Finehout et al. 2004; Dayon et al. 2008) that identifies more than 2500 proteins in CSF of human but specific handling is required.
In Parkinson's disease, limited brain structures are affected by degeneration, hence it is difficult to find a potential biomarker where proteins are of very low abundance in the sample e.g.; α-SYN, DJ-1.
Recent advances in CSF
The concentrations altered α-SYN is specific biomarkers. DJ-1 is also a protein of rare mutation, combining the assays of all CSF proteins such as α-SYN, DJ-1, β-amyloid peptide, Ft3ligand, tau protein (Phosphorylated) together yielded differentiation of Parkinson's disease. Some group of researchers added promising protein biomarkers of CSF leading to the valuable insights of Parkinson's disease.
PROTEIN BASED BIOMARKERS IN CSF
α-synuclein: lewy bodies are mainly composed of fibrillary α-synuclein, which is enriched in neurites. Along with brain cells, it is also seen in nerves, innervating gut, heart and prostate (lwanaga et al. 1999). Some of the immuno reactivity observed in autopsy tissue made α-SYN an excellent testing candidate in Parkinson's disease (Ikemura et al. 2008).
But α-SYN mechanism that is responsible for aggregation still remains unknown Papachroni et al. studied on α-SYN, β-SYN and γ-SYN of patients with sporadic familial Parkinson's disease and found antagonist prevalent to SYN's.
In study carried out by Yaramandra et al. sera of individual suffering from Parkinson's disease constitute antibodies against α-SYN monomers when compared to that of the controls.
DJ-1: PARK 7 or DJ-1 is rare mutations that cause autosomal recessive forms in Parkinson's disease (Bonifati et al. 2003). Some of the major roles played by DJ-1 in Parkinson's disease pathogenesis implicates on transcriptional regulation, anti oxidant protein and as regulator of degradation pathways of protein (Kahle et al. 2009). DJ-1 protein levels measurement in body fluids is a promising biomarker of Parkinson's disease where it is detected in CSF. In Parkinson's disease patient, the level of DJ-1 protein varies throughout the course of Parkinson's disease (Waragai et al. 2008).
CONCLUSION AND FUTURE DIRECTIONS
Several factors to be considered to increase the potentiality of the CSF based protein biomarkers as there is yet no biomarker that is fully validated for Parkinson's disease. Recent advances in proteomic analysis applied to CSF proteins such as α-SYN and DJ-1 have shown profound results as early biomarkers for Parkinson's disease. Based on the assay simplicity, it suggests that high-throughput screening may be possible in long term for the identification of Parkinson's disease progression by tracking metabolic pathways, mitochondrial dysfunction and also by post translation modifications. The advancement in bioinformatics is also an added advantage by combining the techniques to genetic and protein profiling methods.